Antibody Phage Display Methods and Protocols - part 4 doc

Plate transfected cells in 96-well microtiter plates 0.1 mL/well in IMDM/10% FBS and 50 µg/mL gentamicin, in serial dilutions, to determine the size of the transfected library see Note 8

10 Repeat steps 7 and 8 for phh3-VL-VH-lib (see Fig 5) and store in aliquots at

–80°C as the original VL–VH library stock

11 Plate an aliquot of the VL–VH library (1 × 109 bacteria for a library of 1 ×

107 members) at high density on LB–CARB–GLU plates Scrape the bacterial colonies and grow in LB–CARB–GLU containing 10 µg/mL tetracycline(LB-CARB-GLU-TET) to an OD600 of 0.5 Superinfect with VCSM13 helper phage at a 20⬊1 ratio of phage⬊bacteria and prepare phage as indexed elsewhere

in this volume Also determine the cfu/pfu ratio by plating serial dilutions

of the phage on LB–CARB plates for phagemid colonies and on B plates(per L: 10 g Bacto-tryptone, 8 g NaCl, 15 g agar) for plaques (a cfu/pfu ratio

≥ 1 is desirable)

12 Carry out positive/negative selection on the phage following appropriate methods

See index for details

13 After positive/negative selection, infect XL1-Blue supercompetent bacteria with the selected phage Plate the infected cells on LB-CARB-GLU plates in serial dilutions to determine the size of the selected library and at high density to recover the library Scrape the bacterial colonies and superinfect a portion of the culture with VCSM13 helper phage to produce phage for immunoassay (e.g., enzyme-linked immunosorbant assay against the poly-Ag target) Store the remainder of the selected library culture in aliquots in LB–CARB–GLU–15%glycerol at –80°C

14 Prepare dsDNA from the selected library and digest with SacI and XhoI.

Gel-purify the 5-kb backbone (see Note 4) Also isolate the 1.8-kb SacI/XhoI fragment from vector no 578 plPEHPl(+) (see Fig 6), the bidirectional mam-

malian lPPl cassette, which carries the mammalian promoter and leader sequences and the mouse Ig µ enhancer Ligation of these fragments will generate phh3-

VL-m-VH-lib

15 Transform phh3-VL-m-VH-lib into supercompetent HB101 cells and plate on LB–CARB plates in serial dilutions to determine the size of the selected library and to ascertain that ≥90% of library members have the correct-size insert (as determined by diagnostic restriction enzyme digestion of selected clones) and at high density to recover the library

16 Prepare DNA from the recovered phh3-VL-m-VH-lib and digest with EcoRI and HindIII Gel-purify the 2.3-kb fragment containing the VL-VH pairs and

the mammalian lPPl cassette, and ligate with the 15.2-kb EcoRI/HindIII

back-bone from the mammalian vector no 577 pMDV-IgG2b This will generate

pM-DV-IgG2b-lib (see Fig 6).

17 Repeat step 15 for pMDV-IgG2b-lib (see Note 6).

18 Prepare DNA from the recovered pMDV-IgG2b-lib and transfect into Sp2/0

mammalian cells (see Note 7).

19 Plate transfected cells in 96-well microtiter plates (0.1 mL/well) in IMDM/10% FBS and 50 µg/mL gentamicin, in serial dilutions, to determine the size of

the transfected library (see Note 8), and at high density, to obtain multiple clones/well (see Note 9) After overnight incubation, add 0.1 mL/well medium

108 Sharon et al.

Fig 6 Transfer of V-region gene pairs between bidirectional phage-display and mammalian expression vectors (partial maps and not to scale) Prokaryotic elements

are as in Fig 5 Mammalian regulatory elements are oval shaped ampr, ampicillin resistance; ori, prokaryotic origin of DNA replication; P, promoter; E, enhancer;

l, leader sequence; ss, splice site; h, human (all other mammalian regulatory elements

are murine)

Polyclonal Antibody Library Construction 109

(IMDM, 20% FBS, 50 µg/mL gentamicin) containing 1/30X HMX Two days later, aspirate one-half the medium from each well and replace with 0.1 mL/well medium containing 1/5X HMX and 10% (v/v) HES Feed by replacement with medium containing 1X HMX when the cell supernatants in the plates turn orange-yellow about 1 wk later.

20 When clones appear in the dense plates, transfer entire library of transfectomas to

a fl ask Use one-half the cells for cryopreservation in several freezing vials Grow the other half of the cells as desired, and purify the Ab library for immunoassay

and further biological characterization (see Note 10) This is the PCAL.

4 Notes

1 Primers for cDNA synthesis and subsequent PCR steps must be designed for every species

2 The low-stringency fi rst PCR (37°C) ensures amplifi cation of a large repertoire

of V-region genes using a limited primer set; nesting of reverse primers in the

fi rst PCR, compared to the cDNA reaction, and in the second PCR, compared

to the fi rst PCR, minimizes amplifi cation of non-V-region sequences Examples

of primer sequences for the mouse are shown in Fig 3 The principles of design

can be adapted with ease to other species of interest

3 The optimal number of cycles is the minimum number that will yield the maximum amount of V-region gene PCR product To determine this, sample small volumes from a test PCR after 10, 15, 20, 25, and so on, cycles forgel analysis, and use the lowest cycle number yielding a strong-staining band (15 cycles in this lab)

4 For backbone preparation, the vector is linearized by cutting with the fi rst effi cient) enzyme, gel-purifi ed and the recovered DNA fragment is then cut with the second enzyme and gel-purifi ed This procedure minimizes the amount of uncut vector in the backbone sample

5 A library size ≥1 × 106 members is desirable for phh3-VH-lib A library size

≥1 × 108 members is desirable for phh3-VL-VH-lib, although at the time of writing, our largest library has comprised 2 × 107 clones

6 A library size ≥10× the size of the poly-Ag-selected library is desirable, to ensure good representation of every member of the selected library

7 Transfection into Sp2/0 cells can be done by electroporation (7) of 2 × 107 Sp2/0 cells in 0.8 mL PBS/cuvet with 10 µg DNA, linearized by prior digestion with

SalI, followed by gel purifi cation Electroporation conditions are 960 µF and

240 V Alternatively, transfection can be achieved by spheroplast fusion (8).

Prepare spheroplasts from about two OD550 of chloramphenicol-treated bacterial culture, and add 13 mL DMEM/sucrose/MgCl2 to a DMEM-washed monolayer

of Sp2/0 cells in a 10-cm tissue culture dish Centrifuge 5 min at 1200g in

appropriate plate carriers, and aspirate the medium Add 4 mL 50% PEG, and

70 s later, dilute the PEG, and gently wash with DMEM Resuspend in complete medium and incubate for 4 h at 37°C, then harvest the cells by scraping To

110 Sharon et al.

avoid expression of more than one pair of HC and LC per transfected cell, electroporation should be done at a limiting DNA concentration that favors integration and expression of a single plasmid molecule Spheroplast fusion should be done at limiting spheroplast number that favors fusion of a single spheroplast; this may contain up to 1000 copies of the same plasmid per mam-malian cell.

8 A transfected library size ≥10× the size of the poly-Ag-selected library is desirable to ensure good representation of every member of the selected library

9 The library of transfected cells is initially plated in 96-well microtiter plates, to allow development of clones in an immobile crossfeeding environment

10 The library can be regenerated by growth from cryopreserved aliquots of the transfection mixture or by retransfection of pMDV-IgG2b-lib

Acknowledgments

We thank Liyan Chen for discussion and Steven Pageau for computer graphics and manuscript preparation This work was supported by grant no AI23909 from the National Institutes of Health to J Sharon Seshi Sompuram and Chiou-Ying Yang have contributed equally to establishment of this method Chiou-Ying Yang was formerly known as Chiou-Ying Y Kao.

References

1 Sharon, J (1998) Basic Immunology Williams & Wilkins, Baltimore, MD.

2 Sarantopoulos, S., Kao, C Y., Den, W., and Sharon, J (1994) A method for linking

VL and VH region genes that allows bulk transfer between vectors for use in

generating polyclonal IgG libraries J Immunol 152, 5344–5351.

3 Den, W., Sompuram, S R., Sarantopoulos, S., and Sharon, J (1999) A bidirectional phage display vector for the selection and mass transfer of polyclonal antibody

libraries J Immunol Methods 222, 45–57.

4 Baecher-Allan, C M., Santora, K., Sarantopoulos, S., Den, W., Sompuram,

S R., Cevallos, A M., et al (1999) Generation of a polyclonal Fab phage display

library to the protozoan parasite Cryptosporidium parvum Combinatorial Chem

High Throughput Screening 2, 299–305.

5 Santora, K E., Sarantopoulos, S., Den, W., Petersen-Mahrt, S., Sompuram,

S R., and Sharon, J (2000) Generation of a polyclonal fab phage display library

to the human breast carcinoma cell line BT-20 Combinatorial Chem High

Throughput Screening 3, 51–57.

6 Sharon, J., Sarantopoulos, S., Den, W., Kao, C.-Y., Baecher-Allan, C M., Santora,

K E., et al (2000) Recombinant polyclonal antibody libraries Combinatorial

Chem High Throughput Screening 3, 185–196.

7 Sharon, J., Gefter, M L., Wysocki, L J., and Margolies, M N (1989) Recurrent somatic mutations in mouse antibodies to p-azophenylarsonate increase affi nity

for hapten J Immunol 142, 596–601.

Polyclonal Antibody Library Construction 111

8 Sharon, J., Gefter, M L., Manser, T., and Ptashne, M (1986) Site-directed mutagenesis of an invariant amino acid residue at the variable-diversity segments

junction of an antibody Proc Natl Acad Sci USA 83, 2628–2631.

9 Kabat, E A., Wu, T T., Perry, H M., Gottesman, K S., and Foeller, C (1991)

Sequences of Proteins of Immunological Interest U.S Department of Health and

Human Services, Bethesda, MD

10 Barbas, C F I., Kang, A S., Lerner, R A., and Benkovic, S J (1991) Assembly

of combinatorial antibody libraries on phage surfaces: the gene III site Proc Natl

Acad Sci USA 88, 7978–7982.

112 Sharon et al.

From: Methods in Molecular Biology, vol 178: Antibody Phage Display: Methods and Protocols

Edited by: P M O’Brien and R Aitken © Humana Press Inc., Totowa, NJ

be devised to drive the proliferation of the clones of interest in vitro In our own studies of the colorectal cancer (CRC)-associated antigen (Ag) CA-Hb3,

a 50-kDa protein that is recognized by monoclonal antibody (MAb), Hb3 (1),

procedures were developed to drive the proliferation of specifi c B cells from the blood of patients, through exposure to Ag in vitro This has enabled generation, through phage display, of recombinant human Abs against CA-Hb3.

4 Lymphocyte separation solution

5 Dulbecco’s modifi ed Eagle’s medium (DMEM) culture medium

6 Fetal bovine serum (FBS) heat-inactivated at 56°C for 30 min

7 Hank’s balanced salt solution (HBSS)

8 Heparin diluted in PBS or heparinized tubes

Ag Stimulation of B-Lymphocytes In Vitro 113

9 Glutaraldehyde (1%).

10 Butanol-1

11 50 mM Carbonate buffer, pH 9.6.

12 1% Bovine serum albumin (BSA) in PBS

13 Antihuman IgG and IgM horseradish peroxidase conjugates

19 Standard reagents for polymerase chain reaction (PCR) (Taq polymerase, buffer,

deoxyribonucleoside triphosphates [dNTPs], primers, and so on)

3 Methods

3.1 Screening for Seropositive Donors

1 In order to drive a secondary immune reaction during in vitro stimulation, enzyme-linked immunosorbent assay (ELISA) assay should be used to select patients with Abs against the given Ag and/or the Ag itself, if possible If samples are negative for Ag and/or Ab, it may still be worthwhile to go ahead with in

vitro stimulation (see Subheading 3.2.).

2 To test for Abs in serum (plasma, if heparin has been used) against CA-Hb3, the

Ag of interest here, culture the cancer cells overnight at 104 cells/well in 100

µL medium in 96-well plates at 37°C and 5% CO2, then fi x cells with 0.24% glutaraldehyde at room temperature for 10 min Alternatively, coat microtiter wells with 100 µL of 10 µg/mL crude butanol extraction (CBE) Ag at 37°C for

2 h then 4°C overnight The Ag is extracted from cells with 2.5% 1-butanol (2)

and diluted in 0.05 M bicarbonate buffer, pH 9.6 (coating buffer).

3 Wash the plates 3 × 3 min with PBS

4 Block wells with 200 µL of 1% BSA in PBS at room temperature for 30 min

5 Incubate each well with 100 µL of serially diluted plasma at 37°C for 2 h

6 Wash 3 × 3 min with PBS

7 Incubate each well with 100 µL of 1⬊2000 diluted anti-human IgG + IgM HRP conjugate in 1% BSA at 37°C for 1 h

8 Wash 3 × 3 min with PBS

9 Incubate each well with 100 µL OPD (1 mg OPD powder in 2 mL PBS containing

1µL 30% H2O2) as HRP substrate at room temperature for 15 min

10 Add 50 µL 2 M sulfuric acid to each well to stop reaction and read absorbance

at 490 nm in a ELISA reader

3.2 Screening for Ag in Patient Sera

1 To test for Ag in blood samples, sandwich or indirect ELISA procedures can be used if MAbs or polyclonal Abs are available

114 Hu

2 To conduct a sandwich ELISA, dilute a mAb against the Ag of interest to 10 µg/mL

in carbonate buffer and add to a 96-well plate for 37°C for 2 h, then 4°Covernight

3 Follow the procedure above (see Subheading 3.1.), except use an HRP labeled

MAb against the Ag of interest in place of the anti-IgG + IgM HRP conjugate

4 To conduct an indirect ELISA, coat serially diluted plasma to a 96-well plate at 37°C for 2 h, then 4°C overnight

5 Wash and incubate wells with 10 µg/mL of a MAb or polyclonal Ab against the

9 Phage libraries are best constructed from patients who are positive for both Ab

and Ag (see Note 3).

3.3 Recovery and Culture of Lymphocytes

1 Sterile plastic tubes and fl asks are used throughout All solutions and reagents are fi ltered through 0.22-µm fi lter

2 Take 10-mL blood samples from either a cancer patient or a patient with another disease of interest Blood should be collected into a tube containing heparin (up

to 50 U/mL blood) or a heparinized tube

3 Dilute the blood sample with 10 mL HBSS

4 Add 6 mL diluted blood sample to the top of 6 mL lymphocyte separation solution in a wide transparent centrifuge tube with a cap

5 Centrifuge at 4°C or room temperature for 15 min at 250g.

6 Carefully pipet out the white layer containing peripheral blood lymphocytes (PBL) into a fresh 50-mL centrifuge tube

7 Resuspend PBL with 20 mL HBSS and centrifuge at room temperature at 100g

for 3 min

8 Gently resuspend PBL pellet again in 20 mL HBSS

9 Count PBL numbers and viable cells using 0.4% trypan blue exclusion assay

(see Note 5), then centrifuge at 100g for 3 min.

10 Gently resuspend PBL with appropriate volume of DMEM supplemented with

50 U/mL ampicillin and 50 µg/mL streptomycin and 15% heat-inactivated FBS

to adjust cell density to 106 cells/mL in a fl ask

11 For in vitro stimulation, add affi nity-purifi ed Ag to a fi nal concentration of

10 µM (10 µM is equal to 0.5 µg/mL CA-Hb3) or CBE Ag (see Notes 1 and 2; 2) Then add rhIL-2 (see Note 6; 3) to a fi nal concentration of 20 U/mL and

PWM to 10 µg/mL into the PBL culture

Ag Stimulation of B-Lymphocytes In Vitro 115

12 Incubate the PBL at 37°C and 5% CO2 for 5 d Do not change the DMEM–15%FBS supplemented with Ag, rhIL-2, and PWM during these 5 d.

13 At d 5, remove and keep old medium and add 10 mL fresh DMEM–15% inactivated FBS, Ag, rhIL-2, PWM, and antibiotics in the same concentrations as

heat-above (see Subheading 3.3., steps 10 and 11) and culture the PBL for 2 d more or until cell colonies and lymphoblast cells form (see Fig 1 and Notes 3 and 7).

14 Collect the PBL, using a cell scraper for extraction of total RNA and/or further purifi cation of mRNA Total RNA samples can be used to assay Ig transcript

levels (see Subheading 3.4.) or for making phage Ab libraries (see Note 4).

3.4 Assay of Ig Transcript Levels

by Reverse Transcriptase (RT)-PCR

1 Collect in vitro stimulated PBL from tissue culture fl asks by scraping with a cell scraper and spin briefl y to remove culture medium

2 Resuspend the PBL in 10 mL PBS and count cell numbers using trypan blue

exclusion assay (see Note 5).

3 Extract total RNA of the PBL with Trizol reagent or other total RNA extraction reagent according to the manufacturer’s instructions In vitro stimulation procedure should increase total RNA content of the PBL and the abundance of

Ig mRNA For example, 10 µg total RNA was extracted from 10 mL peripheral blood from a colon cancer patient without in vitro stimulation, but 25 µg total RNA was extracted from 10 mL peripheral blood from the same patient (number 1

in Table 1) after in vitro stimulation.

Fig 1 Typical cellular morphology of PBL from colon cancer patient no 1 from Table 1

at d 7 after in vitro stimulation with a colorectal cancer-associated CA-Hb3 Ag

116 Hu

4 To synthesize complementary DNA (cDNA) from total RNAs from the stimulated and unstimulated PBLs, add 1 µg total RNA to 0.2 µg oligo(dT), 10 U RNase

inhibitor, 5 mM dNTPs, 1X RT buffer and 5 U avian myeloblastosis virus RT in a

reaction volume of 20 µL Incubate the reaction tubes at 42°C for 60 min

5 To amplify VH–CH1 (λ) and VL–CL (κ), a touchdown PCR procedure was used

(4) The 5′ primer for amplifi cation of VH–CH1 is 5′-GAGGTGCAGCTGKTGSAGTCTGS-3′, 3′ primer is 5′-GTCCACCTTGGTGTTGCTGGGCTT-3′ For amplifi cation of VL–CL, 5′ primer is 5′-GAWRTTGTGMTGACKCAGTCTCC-3′and 3′ primer is 5′-AGACTCTCCCCTGTTGAAGCTCTT-3′, where R is A or

G, W is A or T, S is C or G, K is T or G β-actin can be used as an internal control (5′-primer is 5′-CTTCTACAATGAGCTGCGTG-3′, and 3′ primer 5′-TCATGAGGTAGTCAGTCAGG-3′) Set up 50-µL PCR reactions con-taining 2 µL cDNA from stimulated or unstimulated PBL, 1X PCR buffer,

200µM of dNTPs, 20 pmol of each 5′-primer or 3′-primer, and 2.5 U Taq DNA

polymerase

6 Amplify with a modifi ed touchdown procedure consisting of three cycles each

of denaturation at 94°C for 30 s, annealing at 55°C for 1 min, and elongation

at 74°C for 1.5 min Repeat for annealing temperatures reduced in steps of 1°C, from 55° to 46°C Follow the touchdown cycles with 10 cycles using an annealing temperature of 45°C and a 10-min extension at 74°C

7 Analyze one-tenth of the PCR reaction by electrophoresis on 1% agarose gels

In our experience, VH–CH1 and VL–CL amplifi cation yields from stimulated PBL were 0.3× greater than from the unstimulated PBL (Fig 2)

4 Notes

1 The use of an affi nity-purifi ed Ag is important since it determines the specifi city

of the phage Abs To make an affi nity column, if the MAb is available, it could

Table 1

Numbers of Total Peripheral Blood Lymphocytes Counted by Trypan Blue Exclusion Assay in 10 mL Peripheral Blood from Four Colorectal Cancer Patients, Before and after In Vitro Stimulation Driven by a

Colorectal Cancer-Associated CA-Hb3 Ag

Total cell no Total cell no

be conjugated with cyanogen bromide-activated Sepharose 4B according to manufacturer’s instructions.

2 If there is no existing MAb against the Ag of interest, crude Ag or recombinant sources of protein or synthetic peptides can be used Because 2.5% 1-butanol

in PBS extracts tumor-specifi c transplantation Ag from cancer cell membranes, CBE Ag extracted in this way from tumor cell lines can be used at a fi nal concentration of 10 µg/mL for in vitro stimulation (2)

3 Successful in vitro stimulation can be judged from the following: morphology changes to cells in culture refl ecting a secondary immune response, specifi cally, the size of PBL and colony formation; the appearance of specifi c Ab against the

Ag of interest in culture supernatant over the 7 d of culture (this can be assessed

by ELISA) (see Subheading 3.1.); the amounts of total RNA from PBL before

and after in vitro stimulation; the yields of PCR products from Ig RT-PCR (see

Subheading 3.3., step 7).

4 For screening of phage Ab libraries, progressive decreases in the concentration of binding Ag are suggested, i.e., use 1 µg/mL affi nity-purifi ed Ag or recombinant protein or synthetic peptide for the fi rst panning, 0.1 µg/mL for the second panning, then 0.01 µg/mL for the third panning step If pure Ag is not available, but a MAb can be obtained, a sandwich procedure can be used for screening To

Fig 2 Assay of Ig transcript levels by RT-PCR The amounts of Ig from the stimulated PBL (VH-CH1 in lane 1 and VL–CL in lane 3) were 0.3× more than those from the unstimulated PBL (VH–CH1 in lane 2 and VL–CL in lane 4) estimated by band brightness β-actin is the internal control The marker (M) was 100 bp DNA ladder (Life Technologies)

118 Hu

carry this out, 1–5µg/mL MAb is coated onto a Petri dish After washing 3 ×

3 min with PBS and blocking with 1% BSA, 50 µg/mL crude Ag (e.g., CBE Ag)

is added to the dish for 1 h at 37°C After washing 3 × 3 min with PBS, the dish

is ready for the fi rst panning; for the second or third screening, concentrations

of the crude Ag can be reduced to 5 or 0.5 µg/mL, respectively If the MAb is not available, crude Ag (50, 5, and 0.5 µg/mL for the fi rst, second, and third screening, respectively) could still be used for screening of phage Ab libraries The step-by-step decreases in Ag concentration may increase the chances of recovering phage clones of high affi nity

5 In my experiments, PBL numbers from 10 mL peripheral blood from a typical colon cancer patient were 1 × 107 before in vitro stimulation and the numbers were 0.98 × 107 7 d later after in vitro stimulation The cell numbers were counted with trypan blue exclusion assay and a hemocytometer, viable cells comprising more than 95% before and after in vitro stimulation After in vitro stimulation, the total numbers of PBLs from 10 mL peripheral blood per patient from four colorectal cancer patients fell to 40–98% of their original numbers

(Table 1).

6 It should be noted that IL-2 alone will induce apoptosis of T-lymphocytes (3).

Therefore, IL-2 and pokeweed mitogen should be added after or simultaneously with Ag

7 After in vitro stimulation, PBLs become rounder and bigger and the classical morphology of a secondary immune response appears In detail, lymphocytes

at d 0 are small and round, lymphoblast-like cells appear at d 3, some colonies form and lymphoblast cells can be observed at d 5, and at d 7, colonies are more

numerous, bigger, and lymphoblast-like cells can still be seen (Fig 1).

References

1 Sun, Q B., Ho, J I L., and Kim, Y S (1986) Human colonic cancer associated

antigens detected by three monoclonal antibodies Chin Med J 99, 63–74.

2 Coggin, J H., Gillis, L D., and Payne, W J., Jr (1984) Differential extraction of tumor-transplantation antigen and embryonic antigen from simian virus 40- and adenovirus 7-induced sarcoma cells of hamsters with 1-butanol and 3 M potassium

chloride J Natl Cancer Inst 72, 853–862

3 Lenardo, M J (1991) Interleukin-2 programs mouse αβ T lymphocytes for

From: Methods in Molecular Biology, vol 178: Antibody Phage Display: Methods and Protocols

Edited by: P M O’Brien and R Aitken © Humana Press Inc., Totowa, NJ

8

The Recovery of Immunoglobulin Sequences

from Single Human B Cells by Clonal Expansion

Ruud M T de Wildt and René M A Hoet

1 Introduction

The development of phage-display technology and the construction of huge libraries of antibody (Ab) fragments have provided an unlimited source of

binders to virtually any antigen (Ag) (1) However, it is unlikely that the heavy

(VH) and light (VL) chains of the Abs obtained from these libraries resemble original in vivo pairings In certain autoimmune diseases and immunological processes, such as B-cell tolerance, these VH and VL combinations can be of crucial importance To be able to determine the original VH and VL combina- tions of Abs, we have set up a single B-cell culture system This comprises the sorting of individual lymphocytes into culture wells using fl ow cytometry, a

culture system to expand these cells (2) and polymerase chain reaction (PCR)

amplifi cation of their variable-region genes, thereby immortalizing the VH and

VL regions from individual human B cells The method relies on the clonal expansion of single B cells in which cell–cell interactions (CD40–CD40L), as well as soluble factors, have been shown to be essential One advantage beyond conventional hybridoma technology is that this method circumvents laborious plating and screening; the advantage compared to phage-display technology

is that original VH and VL pairings can be isolated This system has been used to analyze VH and VL pairings of human immunoglobulin G+ (IgG+)

B cells of unknown specifi city (3) and, combined with a selection on the Ag

U1A, a frequent autoantigenic protein target in patients with systemic lupus

erythematosus, to analyze pairings in Ag-specifi c B cells (4) The effi ciency of

the system makes it possible to analyze large numbers of B cells and should therefore allow rare B-cell activities to be studied.

Recovery of Immunoglobulin Sequences 121

Other technologies that retain original VH/VL pairings involve PCR assembly

of VH and VL within a single cell (5), which has been achieved with

hybrid-omas but has yet to be routinely applied to populations of B cells because of technical problems Others have isolated single Ag-specifi c lymphocytes using

micromanipulation of lymphocytes bound to Ag-coated erythrocytes (6) or Ag-coated beads (7) The VH and VL genes from these single cells are amplifi ed using reverse transcriptase (RT)-PCR, and cloned as functional Ab fragments However, these techniques involve laborious manipulation of every cell of interest and hence suffer low throughput.

2 Materials

2.1 Preparation of Lymphocytes

1 Heparinized blood from a patient group of interest

2 Phosphate-buffered saline (PBS) with and without 0.3% Na citrate

3 Ficoll-Paque (Pharmacia Biotech)

4 Dulbecco’s modified Eagle’s medium (DMEM)–HAM’s F12 (1⬊1) (Gibco product code 21331)

5 Supplemented calf serum (CS) (Hyclone product code A 2151L)

6 Dimethyl sulfoxide

7 Fetal calf serum (Gibco)

8 Fluorescein isothiocyanate (FITC)-conjugated anti-human IgG (Kallestadt, Amiter, TA)

9 Phycoerythryin-conjugated anti-CD19 (Dako)

10 Coulter Epics Elite fl ow cytometer equipped with an automatic deposit unit (Coulter, Hialeah, FL)

11 Target Ag of interest (e.g., U1A)

12 6-Well culture plates (Greiner)

13 0.1 M NaHCO3, pH 9.6

14 Tissue culture incubator with associated gas supply

15 Trypsin (Gibco)

16 Ethylenediamine tetraacetic acid (EDTA)

17 FITC-conjugated anti-CD19 and anti-CD20 monoclonal antibodies (MAbs) (Dako)

2.2 Culture of B Cells

1 96-Well round-bottomed plates (Costar)

2 Phytohemagglutinin (Murex)

3 β-Phorbol-12-myristate-13-acetate (PMA) (Sigma)

4 Freshly cultured EL4-B5 thymoma cells obtainable from R Zubler (see

Note 4).

122 de Wildt and Hoet

2.3 Enzyme-Linked Immunosorbant Assay (ELISA)-Testing

of Culture Supernatant

1 96-Well plates (Nunc, Maxisorp)

2 0.1 M NaHCO3, pH 9.2 or pH 9.6, depending on application (see Subheading

3.3., step 1).

3 Anti-human IgG, IgM, and total Ig (Dako)

4 2% Skimmed milk powder in PBS (PBSM)

5 Tween-20 in PBS (PBST)

6 Horseradish-peroxidase conjugated anti-human IgG, IgM, and total Ig (Dako)

7 PBSM containing 2% CS

8 Substrate solution: 100 mM sodium acetate (NaAc), pH 6.0, containing 100 µg/mL

3′3′5′5′-tetramethylbenzidine and 0.5 µL/mL 30% hydrogen peroxide solution Add the hydrogen peroxide solution immediately before use of the substrate solution

9 1 M Sulphuric acid.

2.4 Cloning of VH/ VL Regions from B-Cell Clones

1 RNAzol (Cinna/Biotecx Laboratories)

2 Chloroform

3 20 mg/mL Glycogen (Boehringer Mannheim) dissolved in Millipore fi ltered H2O

4 Ethanol–NaAc mix: combine 96 mL absolute ethanol with 4 mL 3 M NaAc,

10 SuperScript II RT (100 U/µL; Gibco)

11 Deoxyribonucleoside triphosphate (dNTP) mix (10 mM each nucleotide).

12 Taq DNA polymerase and 10X reaction buffer.

13 QIAquick PCR purifi cation kit (Qiagen, CA)

14 Phage-display or expression vector (e.g., pHENIX)

15 Mouse MAb P5D4 (Boehringer Mannheim)

16 Electrocompetent Escherichia coli TG1 and electroporation apparatus.

17 TYE agar plates: 15 g Bacto-agar, 8 g Na chloride, 10 g tryptone, 5 g yeast extract in 1 L

18 Ampicillin

19 20% Glucose

20 2TY: 16 g tryptone, 10 g yeast extract, and 5 g Na chloride in 1 L

Recovery of Immunoglobulin Sequences 123

21 Isopropyl thiogalactopyranoside (IPTG).

22 Extraction buffer: 200 mM Na borate, pH 8.0, 160 mM NaCl, 1 mM EDTA.

23 Rabbit anti-mouse Ig horseradish peroxidase conjugate (Dako)

1 Dilute heparinized blood with an equal amount of PBS–0.3% Na citrate

Care-fully layer 20–30 mL diluted blood onto 15 mL Ficoll-Paque Centrifuge at 500g

for 30 min at room temperature

2 Remove the layer containing the peripheral blood mononuclear cells (PBMC), transfer to another tube and add at least 3 vol DMEM–HAM’s F12 containing 10% CS

3 Centrifuge the cells for 10 min at 200g, resuspend in DMEM–HAM’s F12–10%

CS, centrifuge, and resuspend in the same medium

4 At this stage, PBMC are either used directly or frozen in culture medium containing 10% dimethylsufl oxide and 50% fetal calf serum

5 When no Ag selection is preferred, PBMC can be used directly to sort single

IgG- or IgM-positive B cells (see Note 8) Label the cells with FITC-conjugated

anti-human IgG and phycoerythryin-conjugated anti-CD19 for 10 min at room temperature at a concentration of 1 µg/106cells Centrifuge the cells for 5 min

at 200g and resuspend in 0.5 mL PBS Viable, single IgG+, CD19+ lymphocytes are then sorted into 96-well plates using a Coulter Epics Elite fl ow cytometer

equipped with an automatic cell deposit unit Continue from Subheading 3.2.

6 When Ag selection is preferred, fi rst remove monocytes from the PBMC by incubating the cells for 1–2 h at 1–2× 106 cells/mL in DMEM–HAM’s F12–10%

CS at 37°C, >98% humidity, and 5% CO2 Recover nonadherent cells for further

selection (see Note 1).

7 Coat 6-well culture plates with of the target Ag (e.g., recombinant U1A) at a concentration of 5 µg/mL in 0.1 M NaHCO3, pH 9.6, overnight at 4°C

8 Wash the coated plates 3× with PBS and add 1–5 × 106 monocyte-depleted PBMC

in 4 mL DMEM–HAM’s F12–10% CS Incubate for 1–2 h at 37°C (see Note 2).

9 Remove unbound cells by washing 6× with PBS Collect those cellsthat have adhered to the target Ag using 300 µL PBS containing 0.05% trypsin,

1.1 mM EDTA Terminate trypsin treatment after 5 min at 37°C by adding 5 mL

DMEM–HAM’s F12–10% CS

124 de Wildt and Hoet

10 Harvest the cells and label with a mixture of anti-CD19 and anti-CD20 MAbs conjugated to FITC for 10 min at room temperature at a concentration of 1 µg/106

cells Centrifuge the cells for 5 min at 200g and resuspend in 0.5 mL PBS.

11 Sort viable, single CD19+/CD20+ cells into 96-well plates using the flow cytometer

3.2 Culture of B Cells

1 First, human T-cell–macrophage supernatant (TSN) is prepared from freshly isolated PBMC (buffycoat) using Ficoll-Paque density centrifugation as described

(see Subheading 3.1., step 1).

2 Wash the cells 3× with DMEM–HAM’s F12–10% CS and culture in the presence

of 5 µg/mL phytohemagglutinin and 10 ng/mL PMA, at a concentration of1.5× 106 cells/mL

3 After 48 h, centrifuge the cell suspension for 10 min at 1000g Harvest the cell

supernatant (TSN) and store in aliquots at –70°C (see Note 3).

4 Single, sorted B cells (see Subheading 3.1., step 5 or Subheading 3.1.,

step 11) are deposited in 96-well plates containing 200 µL/well DMEM–HAM’sF12–10–15% TSN–10% CS and 20,000 irradiated (2500 rad) EL4-B5 thymoma

cells/well (see Notes 3–5).

5 Remove 100 µL from each well at d 3 and 6 and replace with DMEM–HAM’sF12–10% TSN–10% CS

6 Test culture supernatants from the B-cell cultures for (Ag-specifi c) Ab production

(see Subheading 3.3.) at d 10 or 11 (see Notes 6–9).

3.3 ELISA-Testing of Culture Supernatant

1 To detect the production and Ag-specifi c Ig, coat 96-well plates with 100 µL/well

of an Ag solution at 1 µg/mL Ag (e.g., recombinant U1A) in 0.1 M NaHCO3,

pH 9.6 Incubate overnight at 4°C To detect the production of IgG, IgM, or total

Ig (see Note 3), coat plates with the same volume of 1 µg/mL anti-human IgG,

IgM, or total Ig in 0.1 M NaHCO3, pH 9.2

2 Block the plates with 200 µL/well PBSM for 2 h at room temperature, then wash 3× with PBS

3 Mix 40 µL culture supernatant with an equal volume of PBSM, add to the coated plates, and incubate for 1 h at room temperature

4 Wash the plates 3× with PBST and 3× with PBS

5 Detect the binding of IgG, IgM, or total Ig by adding 100 µL/well of the responding horseradish peroxidase conjugated anti-human Ab, diluted 1⬊5000

cor-in PBSM contacor-incor-ing 2% CS Dilute the conjugates 1⬊1000 for detection of Ag-specifi c Ab production

6 Wash the plates 3× with PBST and 3× with PBS

Recovery of Immunoglobulin Sequences 125

7 Add 100 µL/well substrate solution Stop the reactions when the color has developed by adding 50 µL/well 1 M sulphuric acid Measure the OD650–OD450

(see Notes 3 and 6)

3.4 Cloning and Expression of VH/ VL Regions from B-Cell Clones ( see Note 10)

1 Using a Pasteur pipet, remove the medium carefully from wells containing IgG+

or Ag-specifi c Ab-producing cells Resuspend all cells in 200 µL RNAzol and transfer to 1.5-mL microcentrifuge tubes Add 20 µL chloroform, mix the tube contents, and incubate 5 min at 4°C Centrifuge the samples in a microcentrifuge for 15 min and collect the aqueous phase

2 Add 2 µL glycogen solution and precipitate the RNA by adding 2 vol ethanol–

NaAc Mix and incubate for 45 min at 4°C Spin the tubes for 15 min, 15,000g

at 4°C Carefully remove the ethanol–NaAc mix without disturbing the RNA pellet Add 0.5 mL 70% ethanol and spin again for 5 min at 4°C Air-dry the RNA and dissolve in 100 µL Millipore-fi ltered H2O containing 20 U RNasin Precipitate the RNA again using ethanol/NaAc and store at –70°C until further

use (see Note 10).

3 Recover, wash, and air-dry the RNA as above (see Subheading 3.4., step 2).

Dissolve in 20 µL Millipore-fi ltered H2O containing 20 U RNasin Use half of the RNA for fi rst-strand cDNA synthesis and store the remainder at –70°C

4 Add 2 µL of 10 pmol/µL oligo(dT) primer and briefl y centrifuge Heat the mixture to 70°C for 5–10 min, then chill on ice to anneal the primer Add 4 µL

RT buffer, 2 µL 0.1 M dithiothreitol, 1 µL (100 U) SuperScript II RT and

2 µL dNTP mix Mix and incubate for 1 h at 42°C Inactivate the RT reaction

by heating for 2 min at 80°C

5 Use 5-µL aliquots of these cDNAs in separate PCRs to amplify VH, Vκ, and

Vλ genes using family-specific 5′ primers and 3′ constant-region primers

(Table 1; see Note 11).

6 To carry out PCRs, add 20 pmol of each primer in 1X Taq reaction buffer containing 1.5 mM MgCl2, 250 µM dNTPs, and 2.5 U Taq polymerase Carry out 25 cycles

of 94°C, 1 min; 55°C, 1 min; and 72°C, 1.5 min (see Note 12).

7 Purify the PCR products using a QIAquick PCR purifi cation kit, following the manufacturer’s protocol

8 At this stage, PCR products can be used for direct sequencing (see Subheading

3.5.), or for cloning as scFv.

9 In a 3′-nested second PCR, use 1 µL of the fi rst PCR product under the same

conditions as described above (see Subheading 3.4., steps 5 and 6) with primers

containing appropriate restriction sites for cloning As 5′ primers, the same

primers as in Table 1 can be used, extended with Sfi I/NcoI restriction sites for

VH primers (8) and ApaL1 restriction sites for Vκ and Vλ primers (9) As 3′

primers for the heavy chains (HC), JH forward primers with a SalI site (10)

are used, and for the light chains (LC), Jκ or Jλ primers containing a NotI site

(8) are used.

126 de Wildt and Hoet

10 Clone HCs and LCs sequentially into a phagemid vector, such as pHENIX (11),

in which a peptide epitope of the vesicular stomatitis virus glycoprotein is fused

at the C-terminus as a tag for detection using mouse MAb, P5D4

11 Electroporate the ligated vector into electrocompetent TG1 and plate onto TYE plates containing 100 µg/mL ampicillin and 1% glucose

12 To determine whether isolated clones are reactive with the Ag of interest in ELISA, pick single colonies into 2TY containing 100 µg/mL ampicillin and 1% glucose and grow overnight at 37°C

Recovery of Immunoglobulin Sequences 127

Table 1

Primers for Amplifying Rearranged Ab V Genes

Vλ7/8Back: CAG (AG)CT GTG GTG AC(CT) CAG GAG

Vλ9/10Back: CAG (CG)C(TA) G(GT)G CTG ACT CAG CCA

IgG1-4CH1For GTC CAC CTT GGT GTT GCT GGG CTT

Sequencing primers

CH1.lib.seq primer GGT GCT CTT GGA GGA GGG TGC

Cκlib.seq CAA CTG CTC ATC AGA TGG CG

forlinkseq GCC ACC TCC GCC TGA ACC